The present invention relates to polypeptides which have cellulolytic activity and which can be derived from fungae; and to polynucleotides which encode the polypeptides and vectors which can express the polypeptides in a host cell. The polypeptides can be used to degrade cellulose. In particular they can be used in the production or processing of food, animal feed, wood pulp, paper and textiles.
Cellulose is a linear polysaccharide of glucose residues connected by beta-1,4 linkages. In nature, cellulose is usually associated with other compounds, such as hemicelluloses or lignin.
Three different classes of enzymatic activity have been shown to be required for the complete degradation of cellulose into glucose, viz. endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) and beta-glucosidases (EC 3.2.1.21).
Cellobiohydrolases attack cellulose either from the reducing or the non-reducing ends of the cellulose polymer and yield cellobiose (a glucose dimer) as the major product. Two types of cellobiohydrolase are known, cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II). CBH I attacks cellulose from the reducing end of the cellulose polymer, and CBH II from the non-reducing end.
Cellobiohydrolase I and II from Trichoderma reesei have been described in EP-B-0137 280 and U.S. Pat. No. 4,894,338, respectively. CBH I has also been identified in Agaricus bisporus, Phanerochaete chrysosporium, Trichoderma viride and Humicola grisea. Depending on both the natural origin and the previous treatment cellulose exists in many varieties differing in crystallinity, fibre composition, fibre length, fibre thickness. For fast and complete degradation of crystalline cellulose CBH and endoglucanases work synergistically (Teeri, TT., TIBTECH May 15, 1997, p. 160-167).
The invention seeks to provide novel polypeptides with CBH activity which can be used to degrade cellulose. Accordingly, the invention provides in a first aspect a polypeptide which comprises the sequence of SEQ ID No:8 (CBH A) or SEQ ID No:10 (CBH B), a sequence substantially homologous to either sequence, or a fragment of any of these sequences.
The first aspect of the invention also provides a polypeptide which is a CBH from Aspergillus.
The first aspect additionally provides a polypeptide which:
(i) has CBH activity;
(ii) has an activity of at least 50% of the maximum activity over the pH range from 3 to 5;
(iii) has an optimum activity at a temperature which is greater than 50xc2x0 C.; and optionally
(iv) does not have a cellulose binding domain or a linker peptide.
A second aspect of the invention provides a polynucleotide which encodes the polypeptide of the first aspect, and a polynucleotide capable of selectively hybridising to SEQ ID No:5, 9 or a complement thereof.
A third aspect of the invention provides a vector which comprises a nucleotide of the second aspect.
A fourth aspect of the invention provides a host cell which comprises a polynucleotide or vector of the second or third aspects.
A fifth aspect of the invention provides a composition comprising a polypeptide of the invention and, for example, one or more enzyme(s).
A sixth aspect of the invention provides a use of the polypeptide or composition of the first or fifth aspects to degrade cellulose.
One of the advantages of the present invention is that cellobiohydrolases may be produced which are free from endoglucanases. Since CBHs, unlike endoglucanases, do not significantly affect fibre strength they are more suitable than crude cellulase complexes for fibre modification in applications were strength is important, such as in textile and paper manufacture and finishing.
Another advantage is that the availability of cloned CBHs and endoglucanases (WO 97/13862) makes it possible to design tailor-made combinations of CBHs and endogucanases for specific applications.
Yet another advantage of the invention is that the polypeptides of the invention have optimum activity at low pH, unlike known cellobiohydrolases. Therefore, the polypeptides of the invention are well suited for use in industrial processes which are performed at low pH.
Polypeptides
The polypeptide of the invention typically has CBH activity. Generally such an activity would be the activity defined by E.C. 3.2.1.91. The term xe2x80x9cCBH activityxe2x80x9d includes the ability to produce cellobiose from cellulose (as a substrate).
The CBH activity may be CBH I-like activity and/or CBH II-like activity. The cellulose which is acted on by the polypeptide of the invention may be crystalline cellulose. The level of CBH activity of the polypeptide of the invention may be the same as, substantially the same as, or higher or lower than the polypeptide shown in SEQ ID No:8 or 10.
The polypeptide of the invention may or may not comprise a cellulose binding domain. Such a domain is often linked to the catalytic domain via a linker peptide in prior art CBH enzymes. Therefore if the polypeptide of the invention does not comprise a cellulose binding domain then generally it will not comprise a linker peptide either.
The polypeptide of the invention may comprise the sequence of SEQ ID No:8 or 10 or a substantially homologous sequence. The polypeptide may be 65% homologous to SEQ ID No:8 or 10, preferably 80% or 90%, and more preferably at least 95% homologous thereto over the length of SEQ ID No:8 or 10.
The polypeptide of the invention may be derived from a fungus, such as a filamentous fungus. Preferably the (filamentous) fungus is of the species Aspergillus oryzae, Aspergillus sojae, Aspergillus nidulans, species from the Aspergillus niger Group (as defined by Raper and Fennell, The Genus Aspergillus, The Williams and Wilkins Company, Baltimore, pp 293-344, 1965), specifically including but not limited to Aspergillus niger, Aspergillus awamori, Aspergillus tubigensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus japonicus and Aspergillus ficuum. 
In the context of the invention the term xe2x80x9cderived fromxe2x80x9d includes polypeptides which naturally occur in or are produced by these fungae and therefore can be obtained from these fungae, or fragments of such polypeptides. The term also includes polypeptides which are substantially homologous (such as at least 65% homologous to SEQ ID No:8 or 10) to these naturally occurring polypeptides of the invention. Thus the invention includes polypeptides which are not naturally occurring.
The polypeptide of the invention may co-purify with an xcex1-arabinofuranosidase (see for e.g. Example 1). The polypeptide of the invention may thus co-elute with an xcex1-arabinofuranosidase. The xcex1-arabinofuranosidase may be detected using the chromogenic substrate 4- methylumbelliferyl-xcex1-L-arabinofuranoside. The xcex1-arabinofuranosidase may be a fungal xcex1-arabinofuranosidase, such as one from an Aspergillus species, e.g. Aspergillus niger. The xcex1-arabinofuranosidase may be one from-any of the genera or species of fungae mentioned above from which the polypeptide of the invention may be derived.
A polypeptide of the invention may comprise:
(a) a polypeptide comprising the sequence of SEQ ID No:8 or 10; or
(b) a polypeptide from Aspergillus which is a CBH or has CBH activity; or
(c) a homologue or an allelic variant of any of the polypeptides of (a) or (b) from a fungus, an Aspergillus species, from Aspergillus niger or from any of the genera or species of fungus mentioned above from which the polypeptide of the invention may be derived; or
(d) a homologue which is at least 65% homologous to (a), (b) or (c).
Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as xe2x80x9chard homologyxe2x80x9d).
The polypeptide of the invention or fragments thereof may be used to identify cellobiohydrolases in other organisms. For example, they may be used for the production of antibodies. The sequence of the polypeptide of SEQ ID No;8 or 10 and of species homologues and allelic variants can be modified to provide polypeptides of the invention.
The modified polypeptide may retain CBH activity.
Polynucleotides
A polynucleotide of the invention may encode or comprise a sequence that encodes a polypeptide of the invention.
A polynucleotide of the invention may be capable of hybridising selectively with the coding sequence of SEQ ID No:5 or 9 or to the sequences complementary to those coding sequences. Selective hybridisation is typically achieved using conditions of medium to high stringency (for example high stringency conditions include 0.2xc3x97SSC at 60xc2x0 C., and low stringency conditions include 2xc3x97SSC at 60xc2x0 C.).
A nucleotide sequence capable of selectively hybridizing to the DNA coding sequence of SEQ ID No:5 or 9 or to the sequences complementary to those coding sequences will generally be at least 65%, preferably at least 80% homologous to the coding sequence of SEQ ID No:5 or 9 or their complements. Polynucleotides of the invention, which are typically provided in isolated and/or purified form, may comprise DNA or RNA. They may also be polynucleotides which include within them synthetic or modified nucleotides. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art.
Polynucleotides such as a DNA polynucleotide and primers according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art.
For the recombinant production of the polypeptide of the invention a DNA sequence of the invention is used for gene amplification and/or exchange of expression signals, such as promoters, secretion signal sequences, in order to allow economic production of the polypeptide of the invention in a suitable homologous or heterologous host cell. A homologous host cell is herein defined as a host cell which is of the same species or which is a variant within the same species as the species from which the DNA sequence is derived.
Suitable host cells are preferably microorganisms like bacteria, or more preferably fungi such as yeasts or filamentous fungi. A preferred yeast host cell for the expression of a DNA sequence encoding the polypeptide of the invention is selected from the group consisting of the genera Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, and Schizosaccharomyces. More preferably a yeast host cell is selected from the group consisting of the species Saccharomyces cerevisiae, Kluyveromyces lactis (also known as Kluyveromyces marxianus var. lactis), Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica,and Schizosaccharomyces pombe.
Most preferred for the expression of a DNA sequence encoding the polypeptide of the invention are, however, filamentous fungal host cells. Preferred filamentous fungal host cells are selected from the group consisting of the genera Aspergillus, Trichoderma, Fusarium, Penicillium, Acremonium, Neurospora, Thermoascus, Myceliophtora, Sporotrichum, Thielavia, and Talaromyces. More preferably a filamentous fungal host cell is selected from the group consisting of the species Aspergillus oyzae, Aspergillus sojae, Aspergillus nidulans, species from the Aspergillus niger Group as defined by Raper and Fennell (1965, In: The Genus Aspergillus, The Williams and Wilkins Company, Baltimore, pp 293-344), specifically including but not limited to Aspergillus niger, Aspergillus awamori, Aspergillus tubigensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus japonicus and Aspergillus ficuum, and further consisting of the species Trichoderma reesei, Fusarium graminearum, Penicillium chrysogenum, Acremonium alabamense, Neurospora crassa,Myceliophtora thermophilum, Sporotrichum cellulophilum, and Thielavia terrestris.
The expression construct can be used for transformation of the host as part of a vector carrying a selectable marker, or the expression construct is co-transformed as a separate molecule together with the vector carrying a selectable marker. Suitable selectable markers which can be used for selection of the transformed host cells are well known to the skilled person 8, 10. Preferred markers include but are not limited to e.g. versatile marker genes that can be used for transformation of most filamentous fungi and yeasts such as acetamidase genes or cDNAs (the amdS genes or cDNAs from A.nidulans, A.oryzae, or A.niger), or genes providing resistance to antibiotics like G418 or hygromycin.
Alternatively, more specific selection markers can be used such as auxotrophic markers which require corresponding mutant host strains: e.g. URA3 (from S.cerevisiae or analogous genes from other yeasts), pyrG (from A.nidulans or A.niger) or argB (from A.nidulans or A.niger). In a more preferred embodiment, the selection marker is deleted from the transformed host cell after introduction of the expression construct in accordance with the methods described in EP-A-0 635 574, so as to obtain transformed host cells capable of producing the polypeptide which are free of selection marker genes.
For most filamentous fungi and yeast, the expression construct is preferably integrated in the genome of the host cell in order to obtain stable transformants. However, for certain yeasts also suitable episomal vector systems are available into which the expression construct can be incorporated for stable and high level expression, examples thereof include vectors derived from the 2xcexc and pKD1 plasmids of Saccharomyces and Kluyveromyces, respectively. In case the expression constructs are integrated in the host cells genome, the constructs are either integrated at random loci in the genome, or at predetermined target loci using homologous recombination, in which case the target loci preferably comprise a highly expressed gene. A highly expressed gene is herein defined as a gene whose mRNA can make up at least 0.5% (w/w) of the total cellular mRNA, e.g. under induced conditions, or alternatively, a gene whose gene product can make up at least 1% (w/w) of the total cellular protein, or, in case of a secreted gene product, can be secreted to a level of at least 0.1 g/l. A number of examples of suitable highly expressed genes is provided herein below.
An expression construct for a given host cell will usually contain the following elements operably linked to each other in a consecutive order from the 5xe2x80x2-end to 3xe2x80x2-end relative to the coding strand of the sequence encoding the polypeptide of the invention: (1) a promoter sequence capable of directing transcription of the DNA sequence encoding the polypeptide in the given host cell, (2) optionally, a signal sequence capable of directing secretion of the polypeptide from the given host cell into the culture medium, (3) the DNA sequence encoding a mature and preferably active form of the polypeptide, and preferably also (4) a transcription termination region (terminator) capable of terminating transcription downstream of the DNA sequence encoding the polypeptide.
A variety of promoters capable of directing transcription in the host cells of the invention is available to the skilled person 8,10. Preferably the promoter sequence is derived from a highly expressed gene as previously defined. Examples of preferred highly expressed genes from which promoters are preferably derived and/or which are comprised in preferred predetermined target loci for integration of expression constructs, include but are not limited to genes encoding glycolytic enzymes such as triose-phosphate isomerases (TPI), glyceraldehyde-phosphate dehydrogenases (GAPDH), phosphoglycerate kinases (PGK), pyruvate kinases (PYK), alcohol dehydrogenases (ADH), as well as genes encoding amylases, glucoamylases, xylanases, cellobiohydrolases, 1-galactosidases, alcohol (methanol) oxidases, elongation factors and ribosomal proteins. Specific examples of suitable highly expressed genes include e.g. the LAC4 gene from Kluyveromyces sp., the methanol oxidase genes (AOX and MOX) from Hansenula and Pichia, respectively, the glucoamylase (glaA) genes from A.niger and A.awamori, the A.oryzae TAKA-amylase gene, the A.nidulans gpdA gene and the T.reesei cellobiohydrolase genes.
Preferably the polypeptide is produced as a secreted protein in which case the polynucleotide sequence encoding a mature form of the polypeptide in the expression construct is operably linked to a polynucleotide sequence encoding a signal sequence. Preferably the signal sequence is native (homologous) to the DNA sequence encoding the polypeptide. Alternatively the signal sequence is foreign (heterologous) to the DNA sequence encoding the polypeptide, in which case the signal sequence is preferably endogenous to the host cell in which the DNA sequence is expressed. Examples of suitable signal sequences for yeast host cells are the signal sequences derived from yeast a-factor genes. Similarly, a suitable signal sequence for filamentous fungal host cells is e.g. a signal sequence derived from a filamentous fungal (gluco)amylase gene, e.g. the A.niger glaA gene.
Downstream of the DNA sequence encoding the polypeptide, the expression construct preferably contains a 3xe2x80x2 untranslated region containing one or more transcription termination sites, also referred to as a terminator. The origin of the terminator is less critical. The terminator can e.g. be native to the DNA sequence encoding the polypeptide. However, preferably a yeast terminator is used in yeast host cells and a filamentous fungal terminator is used in filamentous fungal host cells. More preferably, the terminator is endogenous to the host cell in which the DNA sequence encoding the polypeptide is expressed.
The recombinant host cells according to the invention may be cultured using procedures known in the art. For each combination of a promoter and a host cell, culture condition are available which are conducive to the expression the DNA sequence encoding the polypeptide. After reaching the desired cell density or titre of the polypeptide the culture may be stopped and the polypeptide may be recovered using known procedures.
In addition, homologues and allelic variants of SEQ ID No:5 or 9 may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridizing to the coding sequence of SEQ ID No:5 or 9 complements.
Selective hybridisation is typically achieved under conditions of medium to high stringency (for example high stringency conditions include 0.2 SSC at 60xc2x0 C., and low stringency conditions include 2xc3x97SSC at 60xc2x0 C.).
Compositions containing the polypeptides
The invention also provides a composition comprising a polypeptide of the invention and, optionally, one or more enzymes. It also provides a composition comprising a recombinant host cell of the invention.
The compositions of the invention may be in a form suitable for packaging and/or storage. In a composition which comprises the polypeptide of the invention the composition is generally of a form in which the CBH activity of the polypeptide is substantially retained. The polypeptide in the composition may be attached to or mixed with a carrier, for example the polypeptide may be immobilized, such as on a solid carrier.
In a composition which comprises host cells of the invention the composition will generally be of a form which allows some or all of the host cells to remain viable. The composition may additionally comprise nutrients for the host cell, which are provided to the host cell when it is cultured.
The composition may be in a form suitable for the process in which it will be used. The composition may be formulated in any convenient way, including as a paste, liquid, emulsion, powder, flakes, granulate, pellet or other extrudate. The composition may be formulated for use in a process to produce human food, animal feed, wood pulp, paper or a textile. It may be stabilised in accordance with methods known in the art.
The composition may comprise additional substances which aid the cellulose degrading activity of the composition. Thus the composition may additionally comprise other enzymes. These other enzymes may be recombinant enzymes, or may have been obtained from an organism in which they occur naturally. The enzymes may have been substantially purified before addition to the composition of the invention, or they may be left substantially unpurified before being added to the composition of the invention.
Non-limiting examples of such other enzymes are endoglucanases (EC 3.2.1.4), other cellobiohydrolases (EC 3.2.1.91) xcex2-glucosidases (EC 3.2.1.21), xylanases, pectinases, mannanases, phytases, alpha-amylase, proteases or various plant cell wall degrading enzymes.
The composition may comprise organisms (e.g. bacteria, fungae or yeast) which produce the above mentioned enzymes.
The composition may additionally comprise (particularly when being formulated for use in animal feed) one or more ionophores, oxidising agents, surfactants, rumen protected amino acids, enzyme enhancers or enzymes which may be produced naturally in the gastro intestinal tract of the animals to be fed.
Uses of the polypeptide, polynucleotide, host cell and composition
The invention provides the use of a polypeptide, host cell or composition of the invention to degrade cellulose. Thus the invention provides a method of degrading cellulose comprising contacting a polypeptide, host cell or composition of the invention with the cellulose (to be degraded).
In the method all or part of the cellulose may be degraded, for example from 0 to 20%, 20 to 40%, 40 to 50%, 50 to 70% or 70 to 100% by weight may be degraded.
The cellulose which is degraded in the method of the invention may be derived from a plant or a microorganism, or may have been synthetically produced by man. The cellulose may be in crystalline form. The cellulose may be partially or wholly derivatised, for example lignified.
The method of degrading cellulose of the invention may be part of a method for producing or processing food or beverages, animal feed, pulp, paper or textiles.
Moreover, the CBHs of the present invention, have broader pH optima than CBHs of the prior art, and are particularly well suited for use in industrial processes which are performed at low pH, where the CBHs of the prior art are less or not active. Since the CBHs are also more thermostable as compared with known CBHs they can be used in a much wider range of application conditions.
In accordance with the present invention, it has been found that the CBHs produced via the present invention may be used in the baking of breads. The incorporation of small amounts of CBH to the flour imparts favourable characteristics to the dough and thus to the bread itself such as an increased loaf volume and better textural characteristics such as break and shred quality and crumb quality.
CBHs either alone or in combination with other pure or crude endoglucanase containing enzyme mixtures may also be added to animal feed compositions which are rich in cellulose. When added to feeds (including silage) for ruminants or monogastric animals (eg. poultry or swine) which feeds contain cereals such as barley, wheat, maize, rye or oats or cereal by-products such as wheat bran or maize bran, or other plant materials such as soy beans and other legumes, the enzyme(s) significantly improve the break-down of plant cell walls which leads to better utilization of the plant nutrients by the animal. As a consequence, growth rate and/or feed conversion are improved.
The CBHs of the invention may also be used for the liquefaction of plant cell wall material, e.g. in the beverage industry.